The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis

<p>Abstract</p> <p>Background</p> <p>Plasmids containing <it>hyl</it>_{<it>Efm </it>}(pHyl_Efm) were previously shown to increase gastrointestinal colonization and lethality of <it>Enterococcus faecium </it>in experimental peritonitis. The <it>hyl</it>_{<it>Efm </it>}gene, predicting a glycosyl hydrolase, has been considered as a virulence determinant of hospital-associated <it>E. faecium</it>, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the <it>hyl</it>_<it>Efm</it>-region and we evaluated their effect on virulence using a murine peritonitis model.</p> <p>Results</p> <p>Five mutants of the <it>hyl</it>_<it>Efm</it>-region of pHyl_EfmTX16from the sequenced endocarditis strain (TX16 [DO]) were obtained using an adaptation of the PheS* system and were evaluated in a commensal strain TX1330RF to which pHyl_EfmTX16was transferred by mating; these include <it>i</it>) deletion of <it>hyl</it>_{<it>Efm </it>}only; <it>ii</it>) deletion of the gene downstream of <it>hyl</it>_{<it>Efm </it>}(<it>down</it>) of unknown function; <it>iii</it>) deletion of <it>hyl</it>_{<it>Efm </it>}plus <it>down</it>; <it>iv</it>) deletion of <it>hyl</it>_<it>Efm</it>-<it>down </it>and two adjacent genes; and <it>v</it>) a 7,534 bp deletion including these four genes plus partial deletion of two others, with replacement by <it>cat</it>. The 7,534 bp deletion did not affect virulence of TX16 in peritonitis but, when pHyl_{EfmTX16Δ7,534}was transferred to the TX1330RF background, the transconjugant was affected in <it>in vitro </it>growth versus TX1330RF(pHyl_EfmTX16) and was attenuated in virulence; however, neither <it>hyl</it>_{<it>Efm </it>}nor <it>hyl</it>_<it>Efm</it>-<it>down </it>restored wild type function. We did not observe any <it>in vivo </it>effect on virulence of the other deletions of the <it>hyl</it>_<it>Efm</it>-region</p> <p>Conclusions</p> <p>The four genes of the <it>hyl</it>_{<it>Efm </it>}region (including <it>hyl</it>_<it>Efm</it>) do not mediate the increased virulence conferred by pHyl_EfmTX16in murine peritonitis. The use of the markerless counterselection system PheS* should facilitate the genetic manipulation of <it>E. faecium </it>in the future.</p

Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase

β-Lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of β-lactamases among pathogenic bacteria limits the efficacy of β-lactam antibiotics. This has created a major public health crisis. The use of β-lactamase inhibitors has proven useful in restoring the activity of β-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-β-lactamases are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the β-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, α3-β7 and β12-α5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics simulations were performed, Markov State Models were built, and convolutional variational autoencoder-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, and R236) were mutated and the activity of the generated L1 variants was evaluated in cell-based experiments. The results demonstrate that there are extremely significant gating interactions between α3-β7 and β12-α5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions

2-Mercaptomethyl-thiazolidines use conserved aromatic-S interactions to achieve broad-range inhibition of metallo-β-lactamases

Infections caused by multidrug resistant (MDR) bacteria are a major public health threat. Carbapenems are among the most potent antimicrobial agents that are commercially available to treat MDR bacteria. Bacterial production of carbapenem-hydrolysing metallo-b-lactamases (MBLs) challenges their safety and efficacy, with subclass B1 MBLs hydrolysing almost all b-lactam antibiotics. MBL inhibitors would fulfil an urgent clinical need by prolonging the lifetime of these life-saving drugs. Here we report the synthesis and activity of a series of 2-mercaptomethyl-thiazolidines (MMTZs), designed to replicate MBL interactions with reaction intermediates or hydrolysis products. MMTZs are potent competitive inhibitors of B1 MBLs in vitro (e.g., Ki ¼ 0.44 mM vs. NDM-1). Crystal structures of MMTZ complexes reveal similar binding patterns to the most clinically important B1 MBLs (NDM-1, VIM-2 and IMP-1), contrasting with previously studied thiol-based MBL inhibitors, such as bisthiazolidines (BTZs) or captopril stereoisomers, which exhibit lower, more variable potencies and multiple binding modes. MMTZ binding involves thiol coordination to the Zn(II) site and extensive hydrophobic interactions, burying the inhibitor more deeply within the active site than D/L-captopril. Unexpectedly, MMTZ binding features a thioether–p interaction with a conserved active-site aromatic residue, consistent with their equipotent inhibition and similar binding to multiple MBLs. MMTZs penetrate multiple Enterobacterales, inhibit NDM-1 in situ, and restore carbapenem potency against clinical isolates expressing B1 MBLs. Based on their inhibitory profile and lack of eukaryotic cell toxicity, MMTZs represent a promising scaffold for MBL inhibitor development. These results also suggest sulphur–p interactions can be exploited for general ligand design in medicinal chemistry

Rational design of benzobisheterocycle metallo-β-lactamase inhibitors:a tricyclic scaffold enhances potency against target enzymes

Antimicrobial resistance is a global public health threat. Metallo-β-lactamases (MBLs) inactivate β-lactam antibiotics, including carbapenems, are disseminating among Gram-negative bacteria, and lack clinically useful inhibitors. The evolving bisthiazolidine (BTZ) scaffold inhibits all three MBL subclasses (B1 - B3). We report design, synthesis and evaluation of BTZ analogs. Structure-activity relationships identified the BTZ thiol as essential, while the carboxylate is replaceable, with its removal enhancing potency by facilitating hydrophobic interactions within the MBL active site. While the introduction of a flexible aromatic ring is neutral or detrimental for inhibition, a rigid (fused) ring generated nM benzobisheterocycle (BBH) inhibitors that potentiated carbapenems against MBL-producing strains. Crystallography of BBH:MBL complexes identified hydrophobic interactions as the basis of potency towards B1 MBLs. These data underscore BTZs as versatile, potent broad spectrum MBL inhibitors (with activity extending to enzymes refractory to other inhibitors), and provide a rational approach to further improve the tricyclic BBH scaffold

Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both L- and D-BTZ enantiomers are micromolar competitive βLIs of all MBL classes in vitro, with Ki sof6-15 μM or 36-84 μM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 μM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the L-BTZ enantiomers exhibit 100-fold lower Ki s (0.26-0.36 μM) than D-BTZs (26-29 μM). Importantly, cell-based time-kill assays show BTZs restore β-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate β-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the L-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. D-BTZ complexes most closely resemble β-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.Fil: Hinchliffe, Philip. University of Bristol; Reino UnidoFil: Gonzalez, Javier Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mojica, María. Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Estados Unidos. Case Western Reserve University; Estados UnidosFil: Gonzalez, Javier Marcelo. Universidad Nacional de Santiago del Estero. Instituto de Bionanotecnología del Noa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Bionanotecnología del Noa; ArgentinaFil: Castillo, Valerie. Universidad de la República; UruguayFil: Saiz Garcia, Cecilia. Universidad de la República; UruguayFil: Kosmopoulou, Magda. University of Bristol; Reino UnidoFil: Tooke, Catherine. University of Bristol; Reino UnidoFil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mahler, Graciela. Universidad de la República; UruguayFil: Bonomo, Robert. Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Estados Unidos. Case Western Reserve University; Estados UnidosFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Spencer, James. University of Bristol; Reino Unid

Boronic Acid Transition State Inhibitors as Potent Inactivators of KPC and CTX-M β-Lactamases: Biochemical and Structural Analyses

Design of novel beta-lactamase inhibitors (BLIs) is one of the currently accepted strategies to combat the threat of cephalosporin and carbapenem resistance in Gram-negative bacteria. Boronic acid transition state inhibitors (BATSIs) are competitive, reversible BLIs that offer promise as novel therapeutic agents. In this study, the activities of two alpha-amido-beta-triazolylethaneboronic acid transition state inhibitors (S02030 and MB_076) targeting representative KPC (KPC-2) and CTX-M (CTX-M-96, a CTX-M-15-type extended-spectrum beta-lactamase [ESBL]) beta-lactamases were evaluated. The 50% inhibitory concentrations (IC(50)s) for both inhibitors were measured in the nanomolar range (2 to 135 nM). For S02030, the k(2)/K for CTX-M-96 (24,000 M-1 s(-1)) was twice the reported value for KPC-2 (12,000 M-1 s(-1)); for MB_076, the k(2)/K values ranged from 1,200 M-1 s(-1) (KPC-2) to 3,900 M-1 s(-1) (CTX-M-96). Crystal structures of KPC-2 with MB_076 (1.38-&amp; ANGS; resolution) and S02030 and the in silico models of CTX-M-96 with these two BATSIs show that interaction in the CTX-M-96-S02030 and CTX-M-96-MB_076 complexes were overall equivalent to that observed for the crystallographic structure of KPC-2-S02030 and KPC-2-MB_076. The tetrahedral interaction surrounding the boron atom from S02030 and MB_076 creates a favorable hydrogen bonding network with S70, S130, N132, N170, and S237. However, the changes from W105 in KPC-2 to Y105 in CTX-M-96 and the missing residue R220 in CTX-M-96 alter the arrangement of the inhibitors in the active site of CTX-M-96, partially explaining the difference in kinetic parameters. The novel BATSI scaffolds studied here advance our understanding of structure-activity relationships (SARs) and illustrate the importance of new approaches to beta-lactamase inhibitor design

The Bifidobacterium dentium Bd1 Genome Sequence Reflects Its Genetic Adaptation to the Human Oral Cavity

Bifidobacteria, one of the relatively dominant components of the human intestinal microbiota, are considered one of the key groups of beneficial intestinal bacteria (probiotic bacteria). However, in addition to health-promoting taxa, the genus Bifidobacterium also includes Bifidobacterium dentium, an opportunistic cariogenic pathogen. The genetic basis for the ability of B. dentium to survive in the oral cavity and contribute to caries development is not understood. The genome of B. dentium Bd1, a strain isolated from dental caries, was sequenced to completion to uncover a single circular 2,636,368 base pair chromosome with 2,143 predicted open reading frames. Annotation of the genome sequence revealed multiple ways in which B. dentium has adapted to the oral environment through specialized nutrient acquisition, defences against antimicrobials, and gene products that increase fitness and competitiveness within the oral niche. B. dentium Bd1 was shown to metabolize a wide variety of carbohydrates, consistent with genome-based predictions, while colonization and persistence factors implicated in tissue adhesion, acid tolerance, and the metabolism of human saliva-derived compounds were also identified. Global transcriptome analysis demonstrated that many of the genes encoding these predicted traits are highly expressed under relevant physiological conditions. This is the first report to identify, through various genomic approaches, specific genetic adaptations of a Bifidobacterium taxon, Bifidobacterium dentium Bd1, to a lifestyle as a cariogenic microorganism in the oral cavity. In silico analysis and comparative genomic hybridization experiments clearly reveal a high level of genome conservation among various B. dentium strains. The data indicate that the genome of this opportunistic cariogen has evolved through a very limited number of horizontal gene acquisition events, highlighting the narrow boundaries that separate commensals from opportunistic pathogens

The urgent need for metallo-β-lactamase inhibitors: an unattended global threat

Due to their superior tolerability and efficacy, β-lactams are the most potent and prescribed class of antibiotics in the clinic. The emergence of resistance to those antibiotics, mainly due to the production of bacterial enzymes called β-lactamases, has been partially solved by the introduction of β-lactamase inhibitors, which restore the activity of otherwise obsolete molecules. This solution is limited because currently available β-lactamase inhibitors only work against serine β-lactamases, whereas metallo-β-lactamases continue to spread, evolve, and confer resistance to all β-lactams, including carbapenems. Furthermore, the increased use of antibiotics to treat secondary bacterial pneumonia in severely sick patients with COVID-19 might exacerbate the problem of antimicrobial resistance. In this Personal View, we summarise the main advances accomplished in this area of research, emphasise the main challenges that need to be solved, and the importance of research on inhibitors for metallo-B-lactamases amidst the current pandemic.Fil: Mojica, Maria F. Case Western Reserve University. School of Medicine. Infectious Diseases Department; United States.Fil: Mojica, Maria F. Veterans Affairs Northeast Ohio Healthcare System. Research Service; United States.Fil: Mojica, Maria F. Universidad El Bosque. Grupo de Resistencia Antimicrobiana y Epidemiología Hospitalaria; Colombia.Fil: Rossi, María Agustina. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Vila, Alejandro J. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Biofísica; Argentina.Fil: Vila, Alejandro J. CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology; United States.Fil: Bonomo, Robert A. Veterans Affairs Northeast Ohio Healthcare System. Research Service; United States.Fil: Bonomo, Robert A. Case Western Reserve University School of Medicine. Department of Medicine, Department of Pharmacology, Department of Molecular Biology and Microbiology, Department of Biochemistry. Case Center for Proteomics and Bioinformatics; United States.Fil: Bonomo, Robert A. Veterans Affairs Northeast Ohio Healthcare System. Medical Service and Geriatric Research Education and Clinical Center; United States.Fil: Bonomo, Robert A. CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology; United States